Friend leukemia virus integration 1 activates the Rho GTPase pathway and is associated with metastasis in breast cancer

Abstract

Breast cancer is the most prevalent malignant disease in women worldwide. In patients with breast cancer, metastasis to distant sites directly determines the survival outcome. However, the molecular mechanism underlying metastasis in breast cancer remains to be defined. In this report, we found that Friend leukemia virus integration 1 (FLI1) proto-oncogene was differentially expressed between the aggressive MDA-MB231 and the non-aggressive MCF-7 breast cancer cells. Congruently, immunohistochemical staining of clinical samples revealed that FLI1 was overexpressed in breast cancers as compared with the adjacent tissues. The abundance of FLI1 protein was strongly correlated with the advanced stage, poor differentiation, and lymph node metastasis in breast cancer patients. Knockdown of FLI1 with small interfering RNAs significantly attenuated the potential of migration and invasion in highly metastatic human breast cancer cells. FLI1 oncoprotein activated the Rho GTPase pathway that is known to play a role in tumor metastasis. This study for the first time identifies FLI1 as a clinically and functionally important target gene of metastasis, providing a rationale for developing FLI1 inhibitors in the treatment of breast cancer.

Keywords: FLI1; RhoA pathway; breast cancer; metastasis; oncogene.

Figure 1. FLI1 expression is associated with metastasis of breast cancer cells

A. Representative RT-PCR data of genes analyzed. Differential expression of growth factors, oncogenes and target genes were compared between the aggressive MDA-MB231 and the non-aggressive MCF-7 cell lines. B. Confirmation of gene expression by quantitative PCR. Data shown are mean ± SEM from three independent experiments by normalization over the β-ACTIN control. **p < 0.01 as compared with MCF-7. C. Western blot of FLI1 in breast cancer cell lines.

Figure 2. Overexpression of FLI1 in breast cancer tissues

A. Immunohistochemical staining of FLI1 in three representative breast cancer samples and their adjacent tissues. Blue square: adjacent normal tissue; red square: carcinoma. B. FLI1 is highly expressed in breast cancer tissues as compared with adjacent normal tissues. C. FLI1 expression scores in breast cancer patients in different clinical stages. D. High FLI1 expression in breast cancer patients with lymph node metastasis. E. Association between lymph node metastasis and FLI1 expression scores.

Figure 3. FLI1 knockdown decreases cell proliferation in two aggressive breast cancer cells

A-B. Knockdown of FLI1 by two siRNAs (siFLI1 1#, siFli1 2#) in MDA-MB231 (A) and MDA-MB453 (B) FLI1 expression was measured by Western blot. siLUC: control siRNA targeting the photinus pyralis luciferase gene. C-D. Inhibition of cell proliferation by FLI1 siRNAs in MDA-MB231 (C) and MDA-MB453 (D) cancer cells.

Figure 4. Analyses of cell cycle and apoptosis after interference of FLI1 expression

A. Cell cycle in MDA-MB231 cells treated with the control siRNA (siluc) and FLI1 siRNA (siFLI1 1#). B. Apoptosis in MDA-MB231 cells following the knockdown of FLI1.

Figure 5. Knockdown of FLI1 inhibits metastasis of breast cancer cells

A. Reduced cell migration in MDA-MB231 and MDA-MB453 cells following the knockdown of FLI1. B. Cell invasion assay. Cells invaded through the collagen-coated membrane of the transwell were counted. All data shown are mean ± SEM from three independent experiments. *p < 0.05 as compared with control cells (siluc). C. Cell adhesion assay. Note the reduced ability of cell adhesion following the knockdown of FLI1.

Figure 6. Activation of the Rho GTPase pathway

A. GST-PBD and GST-RBD fusion proteins used for quantitate the active RhoA and Rac1. B. Detection of the active Rho GTPases by the GST-PBD and GST-RBD pull-down. PBD: the p21-binding domain (PBD) of the Rac1 effector p21-activated kinases (PAK); RBD: the RhoA binding domain (RBD) of the RhoA effector rhotekin. Note the activation of RhoA, Rac1, and CDC42 in FLI1/HA-expressing breast cancer cells (lane 2) as compared with that in the HA control (lane 1). C. Upregulation of RhoGDI in the aggressive MDA-MB231 breast cancer cells. D. Knockdown of FLI1 reduces the Rho GDP-dissociation inhibitor (RhoGDI). E. Activation of the AKT/ERK cascade. Knockdown of FLI1 decreases the activity of the AKT pathway. F. The proposed model of the FLI1/Rho/AKT pathway.